Human mitochondrial DNA sequence comparisons have been made, resulting in the detection of high frequencies of multiple (repeated) nucleotide substitutions and insertions/deletions. Two substitution biases are apparent, one favoring transitions by a factor of 32:1 over transversions, and the other favoring a high rate of turnover of purines relative to pyrimidines on the heavy strand of mtDNA. Their occurrence in coding and non-coding regions as well as rRNA and tRNA genes suggests that these phenomena may result from biases in the mutational pathways. We have also modeled the dynamics of the substitution process in mammalian mtDNA using the information revealed by these and other comparisons. The results of these models make specific predictions about the substitution process. In addition, the models support the hypothesis that while a portion of the mtDNA molecule is relatively free to change at a very rapid rate, the majority of the molecule has very strong selective constraints on base substitutions and other types of sequence alterations.